"Exotic" seeds from Southern Africa as potential Novel Foods? - Chemical composition of manketti nuts (Schinziophyton rautanenii) and ushivi beans (Guibourtia coleosperma).

Various species of Southern African plants and their edible seeds have gained huge importance due to positive health aspects, and there is increasing interest to introduce such seeds as Novel Food on the international market. Especially the seeds of Schinziophyton rautanenii (manketti) and Guibourtia coleosperma (ushivi) could have great potential as a food and food ingredient. Hence, extensive analyses on the chemical composition of manketti nuts and ushivi beans including the analysis of total solids, protein and fat content, soluble carbohydrates, ash, total and free amino acids, biogenic amines and polyamines, fatty acid profile as well as the content of certain B-vitamins and tocopherols were performed. Results obtained showed a valuable nutritional composition, e.g., a true protein content of 22.6% with a ratio of essential amino acids to total amino acid composition of 48% in manketti nuts, while ushivi beans had a true protein content of 8.2% with a similar ratio of essential to total amino acids (45%). Lipid content was 54.1% in manketti nuts, ushivi beans had a value of 7.7%. In both, linoleic acid was the most abundant. Furthermore, ushivi beans had high amounts of vitamin B1 and B2.

"A2 milk" authentication using isoelectric focusing and different PCR techniques.

Genetic variants of milk proteins have attracted great interest for decades as they are related to important issues such as the composition and technological properties of milk. More recently, an "A1/A2 hypothesis" was developed saying that ß-casein variant A1 may be a dietary risk factor for cardiovascular diseases, type 1 diabetes, sudden infant death syndrome and neurological disorders due to the release of ß-casomorphin-7, whereas no evidence for such adverse effects was assumed for ß-casein A2. Thus, the aim of this study was to adapt and establish analytical methods for the identification of genetic variants of ß-casein using isoelectric focusing of milk proteins as well as appropriate PCR techniques. Allele-specific polymerase chain reaction (AS-PCR) proved to be a reliable method for differentiating most common ß-casein variants (A1, A2, B, C), amplification-created restriction site (ACRS)-PCR using three different restriction enzymes allowed also the detection of variant A3, and the restriction fragment length polymorphism (RFLP)-PCR method enabled the reliable discrimination between A2 (homozygote/heterozygote) and non-A2 animals. Since traces of ß-casein A1 were also found in commercial "A2 milk" in Austria, the authentication of such expensive dairy products is urgently recommended, both by genotyping of all dairy cows at farm level (to confirm that all cows are homozygous ß-casein A2A2) and by screening commercial products on the market (to confirm the absence of ß-casein variants A1, B, and C in dairy products labelled "A2 milk") to protect consumers from this unexpected fraud.

Development and validation of a real-time PCR assay to detect Cannabis sativa in food.

Regarding the prospective investigation of food authenticity and adulteration the aim of the present study was the development and validation of a real-time PCR assay to identify hemp (Cannabis sativa) which has gained increasing importance as a valuable food ingredient. The assay targets a specific spacer DNA sequence in Cannabis sativa chloroplasts and detects 1.5 pg hemp DNA, which is equivalent to 18 copies/µL. Corresponding to the very low LOD (0.00031 ng/µL) the method allows the detection of hemp even in the infinitesimal concentration of contaminants. Due to a SNP in position 603, hemp can be identified unequivocally and discriminated from its closest relative hops (Humulus lupulus). The PCR method shows no cross-reactivity with 39 of 46 tested plant species. Low cross-reactivity with mulberry, stinging nettle, lavender, cornflower, wine, figs and hops can be neglected, because the Δ Ct-values are > 14, and the obtained Ct-values are beyond the cut-off for a positive assessment (Ct-values ≤ 33). Moreover, the suitability of the method to identify hemp as a food ingredient was proved by analysing diverse food products such as chocolate or cookies.

A novel UHPLC method for determining the degree of coffee roasting by analysis of furans.

The aim of this study was to develop a multi-analyte UHPLC method for furans and to apply it to commercial coffee samples as well as commercial roasting trials. Furans, as rising time-temperature indicators (TTIs), promised to be an alternative to unsatisfactory roasting temperature measurements. Consequently, a UHPLC-UV method for the determination of 5-hydroxymethyl-2-furfural (HMF), 5-hydroxymethyl-2-furoic acid (HMFA), 2-furfural (F), 5-methylfurfural (MF), 2-furyl methyl ketone (FMC), 2-furoic acid (FA), and for 3-caffeoylquinic acid (3-CQA) was developed and validated. Commercial roasted coffee beans contained 77.7-322 mg/kg HMF, 73.3-158 mg/kg HMFA, 109-200 mg/kg 2-F, 157-209 mg/kg MF, 12.3-32.8 mg/kg FMC, and 137-205 mg/kg FA. Roasting trial samples showed strong rising HMF contents (max.: Arabica: 769 mg/kg, Robusta: 364 mg/kg) followed by a distinct decline. Only MF and FA appeared as steady rising TTIs in the roasting process in Arabica and Robusta beans. 3-CQA fitted well as a decreasing TTI as expected.

High-throughput quantitation of bovine milk proteins and discrimination of commercial milk types by external cavity-quantum cascade laser spectroscopy and chemometrics.

Analysis of bovine milk proteins is crucial in many food and non-food industrial applications, nevertheless labour-intensive wet-chemical, low-throughput methods are still routinely used. In this work, external cavity-quantum cascade laser (EC-QCL) mid-infrared spectroscopy is employed as a rapid method for protein analysis of commercial bovine milk. Combined analysis of the amide I and II bands enabled quantitation of individual proteins (casein, ß-lactoglobulin, α-lactalbumin) and total protein content. IR spectra of spiked and diluted milk samples were employed for calibration of the target analytes in the presence of a complex matrix by partial least squares (PLS) regression modelling. A sample set of different milk types (pasteurized; differently processed extended shelf life, ESL; ultra-high temperature, UHT) was analysed, and results agreed well with reference methods. Quantitation of temperature sensitive proteins enables detailed distinction between milk types experiencing different heat loads during processing, and discrimination between diverse bovine milk types is successfully demonstrated.

Determination of vitamin B12 in four edible insect species by immunoaffinity and ultra-high performance liquid chromatography.

Insects are rich in major nutrients, such as protein and fat. Recently, minor nutrients like vitamins have become the subjects of interest in insects. Hence, this study reports on the development and validation of a method for the determination of vitamin B12 in mealworm (Tenebrio molitor larvae), cricket (Gryllus assimilis), grasshopper (Locusta migratoria) and cockroach (Shelfordella lateralis), using an ultra-high performance liquid chromatography approach with preliminary immunoaffinity chromatography sample preparation. The method was validated regarding linearity, specificity, accuracy and precision, as well as limits of detection/quantification, and was found to be satisfactory for the desired application. Found levels of vitamin B12 were 1.08 µg/100 g for mealworm, 2.88 µg/100 g for cricket, 0.84 µg/100 g for grasshopper, and 13.2 µg/100 g dry weight for cockroach, representing the first validated report on the content of vitamin B12 in edible insects. Observed interferences are likely caused by the presence of pseudovitamin B12.

Fast quantification of bovine milk proteins employing external cavity-quantum cascade laser spectroscopy.

Analysis of proteins in bovine milk is usually tackled by time-consuming analytical approaches involving wet-chemical, multi-step sample clean-up procedures. The use of external cavity-quantum cascade laser (EC-QCL) based IR spectroscopy was evaluated as an alternative screening tool for direct and simultaneous quantification of individual proteins (i.e. casein and ß-lactoglobulin) and total protein content in commercial bovine milk samples. Mid-IR spectra of protein standard mixtures were used for building partial least squares (PLS) regression models. A sample set comprising different milk types (pasteurized; differently processed extended shelf life, ESL; ultra-high temperature, UHT) was analysed and results were compared to reference methods. Concentration values of the QCL-IR spectroscopy approach obtained within several minutes are in good agreement with reference methods involving multiple sample preparation steps. The potential application as a fast screening method for estimating the heat load applied to liquid milk is demonstrated.

Determination of the native forms of vitamin B1 in bovine milk using a fast and simplified UHPLC method.

The aim of this study was to develop a high-throughput UHPLC method for the determination vitamin B1 active compounds; thiamin, thiamin monophosphate and thiamin diphosphate in bovine milk. In order to sustain the native vitamin B1 phosphorus esters, sample preparation is crucial. Various acids as well as commonly used enzymes and their enzyme mixtures were compared. Method accuracy was confirmed using certified reference material as well as comparison with the corresponding CEN method, and was found to be satisfactory. Studied milk samples showed significant amounts of thiamin monophosphate, which can make up to 53.9% of the total vitamin B1 content in commercial milk, and up to 78% in raw milk. Moreover, a tremendous variation of the total content of vitamin B1 was observed between single cows, which ranged from 0.24mg/L up to 0.54mg/L of total vitamin B1.

Analytical assessment of the intense heat load of whipping cream, coffee cream, and condensed milk at retail in Austria and Germany.

Time temperature integrators (TTIs) are useful tools in estimating the heat load applied on differently processed dairy products. The objective of this study was to analyze and assess three TTIs - lactulose, furosine, and acid-soluble ß-lactoglobulin (ß-Lg) - in 70 high heated dairy products at retail in Austria and Germany comprising whipping cream, coffee cream/milk, and condensed milk products. While ß-Lg was not appropriate to evaluate the heat load of these products, furosine and especially lactulose increased with rising intensity of heat treatment, and are appropriate to distinguish between several heating categories analyzed. Pasteurized (n = 8) and "heat treated" (n = 5) whipping cream samples showed lowest furosine (48 ± 14/ 45 ± 19 mg.100 g-1 protein) and low lactulose (29 ± 10/57 ± 28 mg.L-1) concentrations, followed by ESL whipping cream (n = 10), ESL coffee cream (n = 1), and UHT whipping cream (n = 10) (furosine = 72 ± 37/71/161 ± 30 mg.100 g-1 protein; lactulose = 56 ± 41/161/195 ± 39 mg.L-1), respectively. Sterilized condensed milk samples (n = 14) showed the highest concentrations of both TTIs and could be clearly separated from UHT treated samples (n = 5) (furosine = 491 ± 196/216 ± 46 mg.100 g-1 protein; lactulose = 1997 ± 658/409 ± 161 mg.L-1), whereas the so-called heat-treated samples (n = 9) had a heat load in between showing an extreme range of variation for both TTIs.

A novel ultra-high performance liquid chromatography method for the rapid determination of ß-lactoglobulin as heat load indicator in commercial milk samples.

The level of undenatured acid-soluble ß-lactoglobulin can be used as an indicator to assess the heat load applied to liquid milk, thus further allowing the discrimination between milk originating from different thermal production processes. In this work, a new UHPLC method for the rapid determination of bovine ß-lactoglobulin in 1.8min only (total runtime 3min) is presented using simple UV detection at 205nm. Separation selectivity for possibly co-eluting other major whey proteins (bovine serum albumin, lactoferrin, α-lactalbumin, immunoglobulin G) was verified, and the method validated for the analysis of liquid milk samples regarding linearity (20-560µg/mL, R(2)>0.99), instrumentation precision (RSDs<2.8%), limits of detection and quantification (7 and 23mg/L milk), repeatability of sample work-up (RSDs≤2.6%) and method recovery (103%). In total, 71 commercial liquid milk samples produced using different preservation techniques (e.g., thermal or mechanical treatment), hence featuring different applied heat loads, were profiled for their intrinsic undenatured acid-soluble ß-lactoglobulin levels. As expected, pasteurized milk showed the highest concentrations clearly above 3000mg/L due to pasteurization being the mildest thermal treatment, while in contrast, ultra-high temperature heated milk featured the lowest amounts (<200mg/L). For extended shelf life (ESL) milk, quite diverse levels were determined ranging from ∼100 up to 4000mg/L, thus clearly illustrating variable applied heat loads and impacts on the "nativeness" of milk essentially due to the fact that the production technologies used for ESL milk may differ significantly, and are currently not regulated in the EU.

Application of UHPLC for the determination of free amino acids in different cheese varieties.

A rapid ultra-high performance liquid chromatography (UHPLC) protocol for the determination of amino acids as their respective 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatives was successfully applied for assessing free amino acid levels in commercial cheese samples representing typical product groups (ripening protocols) in cheesemaking. Based on the Waters AccQ.Tag™ method as a high performance liquid chromatography (HPLC) amino acid solution designed for hydrolyzate analyses, method adaptation onto UHPLC was performed, and detection of AQC derivatives was changed from former fluorescence (λ(Ex) 250 nm/λ(Em) 395 nm) to UV (254 nm). Compared to the original HPLC method, UHPLC proved to be superior by facilitating excellent separations of 18 amino acids within 12 min only, thus demonstrating significantly shortened runtimes (>35 min for HPLC) while retaining the original separation chemistry and amino acid elution pattern. Free amino acid levels of the analyzed cheese samples showed a high extent of variability depending on the cheese type, with highest total amounts found for original Italian extra-hard cheeses (up to 9,000 mg/100 g) and lowest for surface mold- or bacterial smear-ripened soft cheeses (200-600 mg/100 g). Despite the intrinsic variability in both total and specific concentrations, the established UHPLC method enabled reliable and interference-free amino acid profiling throughout all cheese types, thus demonstrating a valuable tool to generate high quality data for the characterization of cheese ripening.

Application of UHPLC for the simultaneous analysis of free amino acids and biogenic amines in ripened acid-curd cheeses.

A new UHPLC method for the simultaneous determination of amino acids and biogenic amines in a single run, and its first application to profile ripened acid-curd cheeses was presented. After pre-column derivatization with 6-aminoquinolyl-N-hydroxy succinimidyl carbamate (AQC), 23 amino acids and 15 amines were separated in 9min only (12min total run time), and eluates monitored using their UV response at 249nm. Limits of detection (0.05-0.29mg/100g) and quantification (0.16-0.97mg/100g), repeatability for sample preparation (1.0-6.1% RSD) and method recoveries (83-120%) were found suitable for cheese analysis. In total, 47 acid-curd cheeses classified into sub-groups like cooked, Quargel-type or grey cheeses were analyzed for their free amino acid and amine (histamine, tyramine, putrescine, cadaverine, and tryptamine) contents, which (as expected) were highlighted by a great variability. Total free amino acid levels ranged between less than 100 and more than 4000mg/100g (median 567mg/100g), implying that for some cheeses less or not ripened/fresh quark was used for production or, in contrast, a higher degree of proteolysis had occurred. For the sum of biogenic amines, median concentration was determined at 7.0mg/100g, while only 5% of all cheeses had levels higher than 161.9mg/100g. Thus, the obtained results suggest quite acceptable biogenic amine levels for (mostly underrated) ripened acid-curd cheeses, although partly exceptional high concentrations (>250mg/100g) were indeed observed in individual samples.

Quantification of cow's milk percentage in dairy products - a myth?

The substitution of ewe's and goat's milk for cheaper cow's milk is still a fraudulent practice in the dairy industry. Moreover, soy-based products (e.g., soy milk, yoghurt) have to be checked for cow's milk as they are an alternative for people suffering from an allergy against bovine milk proteins. This work reports the evaluation of different protein-based electrophoretic methods and DNA-based techniques for the qualitative detection as well as the quantitative determination of cow's milk percentage in dairy and soy milk products. Isoelectric focusing (IEF) of γ-caseins using an optimized pH gradient was appropriate not only for the detection of cow's milk, but also for an estimation of cow's milk percentage in mixed-milk cheese varieties. Urea-polyacrylamide gel electrophoresis (PAGE) proved the method of choice to detect cow's milk in soy milk products, whereas IEF and SDS-PAGE of proteins were not applicable due to false-positive results. Polymerase chain reaction (PCR) analysis was used to confirm the results of protein-based electrophoretic methods. Problems inherent in quantitative analysis of cow's milk percentage using protein-based techniques and even more using DNA-based methods were emphasized. Applicability of quantitative real-time PCR for the determination of cow's milk percentage in mixed-milk cheese was shown to be hampered by several factors (e.g., somatic cell count of milk; technological parameters influencing the final DNA concentration in ripened commercial cheese samples). The implementation of certified reference standards (of major relevant cheese groups) containing 50% cow's milk was urgently recommended to enable at least a yes/no decision in commercial mixed-milk cheese samples.

Diversity of the resident microbiota in a thermophilic municipal biogas plant.

Biogas plants continuously convert biological wastes mainly into a mixture of methane, CO2 and H2O-a conversion that is carried out by a consortium of bacteria and archaea. Especially in the municipal plants dedicated towards waste treatment, the reactor feed may vary considerably, exposing the resident microbiota to a changing variety of substrates. To evaluate how and if such changes influence the microbiology, an established biogas plant (6,600 m3, up to 600 m3 biogas per h) was followed over the course of more than 2 years via polymerase chain reaction-denaturing gradient gel electrophoresis of 16S rRNA genes and subsequent sequencing. Both the bacterial and the archaeal community remained stable over the investigation. Of the bacterial consortium, about half of the sequences were in decreasing order of occurrence: Thermoacetogenium sp., Anaerobaculum mobile, Clostridium ultunense, Petrotoga sp., Lactobacillus hammesii, Butyrivibrio sp., Syntrophococcus sucromutans, Olsenella sp., Tepidanaerobacter sp., Sporanaerobacter acetigenes, Pseudoramibacter alactolyticus, Lactobacillus fuchuensis or Lactobacillus sakei, Lactobacillus parabrevis or Lactobacillus spicheri and Enterococcus faecalis. The other half matched closely to ones from similar habitats (thermophilic anaerobic methanogenic digestion). The archaea consisted of Methanobrevibacter sp., Methanoculleus bourgensis, Methanosphaera stadtmanae, Methanimicrococcus blatticola and uncultured Methanomicrobiales. The role of these species in methane production is discussed.

Antibiotic susceptibility testing of Bifidobacterium thermophilum and Bifidobacterium pseudolongum strains: broth microdilution vs. agar disc diffusion assay.

There is urgent need for having available suitable methods and data regarding the susceptibility levels of antibiotic resistant and sensitive strains of bifidobacteria. Based on a defined standard operation procedure, agar disc diffusion and broth microdilution were compared in order to evaluate the antimicrobial susceptibility profiles of 82 B. pseudolongum and 80 B. thermophilum strains mainly originating from the meat production chain. The methods that were assessed showed interpretable agreement within this study. The disc diffusion zone diameters are highly reproducible making the method a useful alternative to broth microdilution for antimicrobial susceptibility screening of bifidobacteria.

Antibiotic susceptibility of Bifidobacterium thermophilum and Bifidobacterium pseudolongum isolates from animal sources.

The widespread use of antimicrobial substances has led to resistant populations of microorganisms in several ecosystems. In animal husbandry, the application of antibiotics has contributed to resistance development in pathogenic and commensal bacteria. These strains or their resistance genes can be spread along several ecological routes, including the food chain. Antibiotic resistance is important in terms of the safety of industrial strains, such as probiotics for food and feed. Bifidobacterium thermophilum and Bifidobacterium pseudolongum are known to comprise the major part of the bifidobacterial microbiota in the gut and feces of cattle and pigs. In this study, the antimicrobial susceptibility in bifidobacterial isolates of these species was investigated. Isolates from the beef and pork production chain were identified and typed to strain level, and the antimicrobial susceptibility level was tested to a set of antibiotics. Isolates with low susceptibility levels were screened by PCR for already described resistance genes. Strains atypically resistant to clindamycin, erythromycin, and tetracycline were determined. The resistance genes tet(O), tet(W), and erm(X) were detected in the bifidobacterial species that were examined.

How safe is safe?--a case of Lactobacillus paracasei ssp. paracasei endocarditis and discussion of the safety of lactic acid bacteria.

We report a case of endocarditis due to Lactobacillus paracasei ssp. paracasei, which could be distinguished from Lactobacillus strains used for the fermentation of dairy products by randomly amplified polymorphic DNA-polymerase chain reaction. The safety of biotechnical lactic acid bacteria use is also discussed.

Methods used for the isolation, enumeration, characterisation and identification of Enterococcus spp. 1. Media for isolation and enumeration.

Due to their significance in food, feed, environmental and clinical samples, the detection and enumeration of enterococci have become an important issue not only in daily routine but also in current research activities. Several media and protocols have been published for diverse purposes, but there is no single method, which universally meets all requirements. Depending on the nature of the accompanying microflora and its level, certain substrates and modifications thereof have to be used, taking into account various drawbacks and advantages. In addition to the historical applications (examination of water, different kinds of foods, intestinal and other clinical specimen), the detection of vancomycin-resistant enterococci (VRE) has become an important task, since VRE have found to be frequently involved in nosocomial infections. Moreover, contradictory methodological recommendations can be found in the literature. This paper will give a systematic survey of the different media and methods proposed during the last two decades. Emphasis is placed on compositional details and on specific applications of the media described.

Methods used for the isolation, enumeration, characterisation and identification of Enterococcus spp. 2. Pheno- and genotypic criteria.

This paper reviews the methodology applied for the identification and characterisation of enterococci and covers phenotypic, genotypic and phylogenetic techniques. Although conventional phenotypic typing schemes are useful for rapid and simple identification of enterococcal species for routine applications, other methods like standardised sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), multilocus enzyme electrophoresis (MLEE), antimicrobial susceptibility testing, serotyping, pyrolysis mass spectrometry (pyMS) and vibrational spectroscopic methods allow a more in-depth characterisation of enterococci. Many of the recently described enterococcal species exhibit deviations from hitherto so-called classical enterococci with regard to their phenotypical properties. Therefore, genotypic methods have to be used to clarify their possible assignment to the genus Enterococcus. In this review, special emphasis is given on recently developed polymerase chain reaction (PCR)-based typing methods such as random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), specific and random amplification (SARA) and modifications of PCR-ribotyping as well as pulsed-field gel electrophoresis (PFGE) and partial sequence analysis. The use of PCR and probes for genus and species identification of enterococci is also considered like the application of sequence data of conserved DNA regions (e.g., ribosomal ribonucleic acid (rRNA) genes) in the case of species identification.

Protein fingerprinting of Saccharomyces isolates with therapeutic relevance using one- and two-dimensional electrophoresis.

In therapeutic products and preparations Saccharomyces cerevisiae is used because of its nutritive properties. Moreover, so called Saccharomyces boulardii yeasts are used in the prevention and treatment of several types of diarrhea. Taxonomically however, S. boulardii is not accepted as a distinct species. The protein fingerprint obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was identical for all isolates and therefore confirmed the designation of S. boulardii to the species S. cerevisiae. In contrast, using native polyacrylamide gel electrophoresis, 12 different protein fingerprints were detected, and allowed grouping of the product isolates. The spot patterns obtained by two-dimensional electrophoresis revealed a large degree of resemblance, however, small qualitative expression differences could be detected as well. Firstly, a spot having an isoelectric point of approximately 6 and 30 kDa could not be detected in S. boulardii yeasts. Secondly, nine different formations of spots occurred in the region around 16 kDa and pH 6. Therefore, on the one hand, it could be demonstrated that all of the product isolates belong to the same species, and on the other hand, it was possible to extensively subdivide the strains. In particular, two-dimensional electrophoresis allowed clustering of so called S. boulardii strains within the species S. cerevisiae.